FR2684378A1 - Process for assaying SOD activity using brazilin or one of its derivatives, necessary for its implementation, compounds derived from brazilin and their preparation - Google Patents

Abstract

The process according to the invention for assaying SOD activity in a liquid medium uses the activation, by SOD activity, of the autoxidation of a reagent of formula: PO3R<6>, R<2> = H, alkyl (1 to 6 C), COR<5>, SO2R<5>, PO2R<5>, CO2R<6>, SO3R<6> or PO3R<6>, R<3> = H or OR<4>, with R<4> = H, alkyl (1 to 6 C), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl, p-carbomethoxybenzyl, vinyl, trimethylsilyl, COR<5>, SO2R<5>, PO2R<5>, CO2R<6>, SO3R <6 , PO3R<6>, COR<8>, CO2R<7>, CON(R<8>,R<9>), NO2, SO2R<8>, PO3R<8> or PO2N(R<8>,R <9>), R<5> = CF3, CF2R<7> or p-nitrophenyl, R<6> = H, CF3, CF2R<7> or p-nitrophenyl, R<7> = alkyl (1 to 6 C), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or p-carbomethoxybenzyl, R<8> = H, alkyl (1 to 6 C), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or p-carbomethoxybenzyl, and R<9> = H, alkyl (1 to 6 C), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or p-carbomethoxybenzyl. Application to assaying SOD activity in a sample, especially a biological sample, in particular using a single measurement and a calibration curve.

Description

Method for assaying the SOD activity using brazilin or one of its derivatives, necessary for its implementation, compounds derived from brazilin and their preparation.

The present invention relates to the assay of superoxide dismutase (SOD) activity.

A more particular subject of the invention is a new method for assaying the SOD activity, in particular in biological samples, using brazilin or one of its derivatives defined below, of the kits or kits for the implementation. implementation of this process and of the new derivatives of brazilin which can be used in this process as well as their preparation.

The tissues of aerobic organisms, and those of the human body in particular, are home to a continuous production of superoxide 2-- ion, which results from the process of cellular respiration, as well as many essential metabolic pathways.

This superoxide production increases sharply when these organisms are subjected to "oxidative stress" of toxicological origin (hyperoxia, irradiation, intoxication by xenobiotics which generate free radicals, in particular) or physiopathological (inflammation, ischemia, post-ischemic reperfusion, in particular. ).

It is accepted by the whole scientific community that the superoxide ion is very toxic, although the mechanisms underlying this toxicity remain controversial (reference 1).

To protect against the deleterious effects of the superoxide ion, aerobic organisms have enzymatic systems, superoxide dismutases (SOD), which catalyze the degradation of the superoxide ion according to the following disproportionation reaction

Given the major protective role of this disproportionation reaction with respect to the toxicity of the superoxide ion, the catalytic activity of intra- and extracellular SODs conditions the survival of the tissues of an aerobic organism (references 2 to 5 ).

A method for assaying SOD activity would be an analytical tool of major interest for biological researchers as well as for clinical chemistry laboratories, provided it is sensitive, specific, rapid and not very complex.

Many methods for assaying SOD activity have been published and criticized (references 6 to 10), but it is generally accepted that there is still no satisfactory compromise between the reliability and the complexity of the existing methods because the measurement of an SOD activity poses two major problems that none of the methods published to date has made it possible to solve in a simple way.

The first problem is that of the spontaneous decomposition of the sole substrate, the superoxide ion, which is very rapid at pH close to 7, and which although strongly slowed down, remains significant at pH 9, pH beyond which certain types natural SODs are quickly inactivated.

Obtaining a sufficiently stable stationary concentration therefore requires the presence of a dynamic source of superoxide in the assay solution.

Electrolytic sources require complex equipment and pre-extraction of the SOD catalyst, which is prohibitive in Clinical Chemistry.

Photochemical sources such as riboflavin lead to methods which cannot be standardized and which are complex to implement.

Enzyme sources such as xanthine oxidase require solutions to be prepared immediately.

They are expensive, a source of artifacts and difficult to automate.

The purely chemical sources are reduced to autoxidizable compounds such as 6-hydroxydopamine, pyrogallol, hydroxylamine or sulfite ion, the autoxidation of which is generally inhibited by SOD.

These chemical reagents rarely give precise and sensitive results on crude biological extracts, and they require the extemporaneous preparation of an anaerobic reagent solution; their use is therefore difficult to automate.

The second problem is due to the fact that it is difficult to directly measure ("direct" assay) the disappearance of the 02 e superoxide substrate - as a function of time, or the appearance of its disproportionation products, oxygenated lattice.
H202 (hydrogen peroxide) and oxygen, taking into account the concentrations and pHs accessible under the conditions of an assay for SOD activity.

The only "direct" tectrophotometric method is that of MARKLUND (references 11 and 12) which measures the speed of disappearance of the superoxide ion at pH 9.5, which does not allow the determination of all natural SODs.

This method requires a fast spectrophotometer. The high concentrations of superoxide and the wavelength (250 nm) used make the method imprecise, forcing the experimenter to take several measurements to obtain an average value. In addition, it is necessary to add catalase to avoid inactivation of the
SOD-Cu / Zn, and a new solution of potassium superoxide must be re-prepared immediately before each assay.

This direct spectrophotometric method is therefore difficult to implement, and it does not seem possible to improve it significantly.

The corollary is that the popular methods, that is to say those which are used most often, are generally indirect: they are based on the competition between the SOD activity that one seeks to measure, and the reaction of l superoxide ion with a chromogenic scavenger such as ferric cytochrome C or tetrazolium nitroblue (NBT), the reaction product of which can be measured by UV / visible spectrophotometry. A chemiluminescent trap, such as luminol or lucigenin, can also be used with greater measurement sensitivity, but problems of interference and instrument availability preclude the use of such methods by a layman laboratory.

In addition to the often insufficient specificity of chromogenic reagents with respect to superoxide, the main drawback of competitive assays is that they require linearization of the inhibition values (by
SOD), on 5 or 6 samples of increasing dilutions.

Finally, a certain number of immunoassays of SOD-Cu / Zn or of SOD-Mn are known.

Some are very sensitive, but they do not provide information on the enzymatic activity of the sample, which depends in particular on the degree of possible inactivation of the enzyme. They are also long to implement and relatively complex.

Measurement artefacts and assay multiplication are therefore intrinsically linked to all spectrophotometric methods accessible to most laboratories, which does not meet the needs of research laboratories and remains incompatible with the standardization requirements of Clinical Chemistry.

If spectrophotometry is used
UV / visible, it seems difficult, if not impossible, to develop an assay method which is at the same time sensitive, more specific, simpler and more reliable than the existing methods, without having recourse to the monitoring in kinetics of a reaction activated by SOD activity rather than that of a reaction inhibited by SOD activity or a "direct" method.

In this regard, a recent method (references 13 and 14), using an autoxidizable dye, hematoxylin, differs from the others insofar as it exploits an activation of the autoxidation of the reagent by the SOD activity.

In fact, hematoxylin autoxidizes at pH> 6.5 (reference 13). The rate of autoxidation increases by a factor close to 100 between pH 7 and 9. SOD inhibits this autoxidation at pH <8.1, while it activates it at pH> 8.1.

This observation has led to the development of an assay, based on spectrophotometric monitoring of the formation of hematein at 560 nm, which is accelerated by SOD at alkaline pH. The implementation of this method is difficult due to the instability of the reagent and it remains dependent on a phase of preparation of the sample (dialysis) to eliminate the interference due to the presence of reducing compounds, such as for example glutathione.

In addition, the instability of the oxidation product measured, haematein, and the low sensitivity of the assay (at pH> 8.1), linked in part to the decomposition of haematein, but also to the presence of two potential oxidation sites (catechols) limit the value and use of this method.

In conclusion, the hematoxylin method, although it is of some interest, has drawbacks which do not allow its general use for the determination of SOD activity, in particular in biological samples.

The present invention aims in particular to - have a reagent which is optionally protected from oxidation outside the measurement conditions, and the autoxidation product of which is stable during the measurement - to increase the sensitivity of the assay with respect to a method using hematoxylin (under conditions of activation of autoxidation) - eliminate the most probable possible interferences such as those linked to the presence of mercaptans such as glutathione, for example - develop a method using a reaction medium with a pH less than or equal to 9, so as to allow an assay of the activity of known SODs, without risk of rapid inactivation.

These aims are achieved according to the invention which is essentially based on the use, instead of hematoxylin, of brazilin or one of its derivatives defined below, the autoxidation products of which are much more stable than haematein, in an assay process whose principle is based on an activation of the autoxidation of these compounds by SOD activity and which makes it possible to have a specific and sensitive measurement of this activity.

The invention also provides novel reagents derived from brazilin which, in the assay medium hydrolyze to brazilin or a derivative thereof and constitute stable chemical reagents outside the measurement conditions.

The invention also provides a process for stabilizing a stock solution of brazilin or one of its derivatives defined below, by means of boron derivatives, in particular boric acid and its derivatives such as 1 ' phenylboric acid.

Finally, according to one of its variants, the invention provides a method which uses a rapid reagent for the thiol groups possibly present in the sample to be assayed, such as in particular a biological sample, which allows d Hz é1 to eliminate interference. due to these thiol groups, in particular those which are due to the presence of glutathione or albumin in the sample.

dans laquelle
R1 = hydrogène, COR5, S02R5, Po2R5, C02R6, S03R6 ou Po3R6,
R2 = hydrogène, alkyle (avec 1 à 6 atomes de carbone), COR5,
So2R5, P02R5, Co2R6, S03R6 ou Po3R6,
R3 : hydrogène ou OR4, avec
R4 = hydrogène, alkyle (avec 1 à 6 atomes de carbone),
benzyle, p-nitrobenzyle, p-diméthylaminobenzyle, p
carbométhoxybenzyle, vinyle, triméthylsilyle, COR5,
S02R5, Po2R5, C02R6, S03R6, P03R6, COR8, C02R7,
CON(R8,R9), N02, S02R8, P53R8 ou P02N(R8,R9),
R5 = CF3, CF2R7 ou p-nitrophényle,
R6 = hydrogène, CF3, CF2R7 ou p-nitrophényle,
R7 = alkyle (avec 1 à 6 atomes de carbone), benzyle, p
nitrobenzyle, p-diméthylaminobenzyle ou p-carbo
méthoxybenzyle,
R8 = hydrogénée, alkyle (avec 1 à 6 atomes de carbone),
benzyle, p-nitrobenzyle, p-diméthylaminobenzyle ou
p-carbométhoxybenzyle, et
R9 = hydrogène, alkyle (avec 1 à 6 atomes de carbonate),
benzyle, p-nitrobenzyle, p-diméthylaminobenzyle ou
p-carbométhoxybenzyle.According to one of its aspects, the subject of the invention is a method for assaying the superoxide dismutase (SOD) activity in a liquid medium, which method uses the activation of the autoxidation of a reagent by the activity
SOD, characterized in that said reagent is a compound corresponding to the general formula I

in which
R1 = hydrogen, COR5, S02R5, Po2R5, C02R6, S03R6 or Po3R6,
R2 = hydrogen, alkyl (with 1 to 6 carbon atoms), COR5,
So2R5, P02R5, Co2R6, S03R6 or Po3R6,
R3: hydrogen or OR4, with
R4 = hydrogen, alkyl (with 1 to 6 carbon atoms),
benzyl, p-nitrobenzyl, p-dimethylaminobenzyl, p
carbomethoxybenzyl, vinyl, trimethylsilyl, COR5,
S02R5, Po2R5, C02R6, S03R6, P03R6, COR8, C02R7,
CON (R8, R9), N02, S02R8, P53R8 or P02N (R8, R9),
R5 = CF3, CF2R7 or p-nitrophenyl,
R6 = hydrogen, CF3, CF2R7 or p-nitrophenyl,
R7 = alkyl (with 1 to 6 carbon atoms), benzyl, p
nitrobenzyl, p-dimethylaminobenzyl or p-carbo
methoxybenzyl,
R8 = hydrogenated, alkyl (with 1 to 6 carbon atoms),
benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or
p-carbomethoxybenzyl, and
R9 = hydrogen, alkyl (with 1 to 6 carbonate atoms),
benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or
p-carbomethoxybenzyl.

In general formula I above, when R1 and
R2 represents hydrogen and R3 represents OR4 with R4 = hydrogen, the compound in question is brazilin which is a commercial product.

The compounds of general formula I other than brazilin (R1 = R2 = hydrogen and R3 = OH) are new.

These compounds can be classified into four series.

The compounds of the first series which correspond to the general formula I, in which
R1 = R2 = hydrogen,
R3 = hydrogen or OR4, with
R4 = alkyl (with 1 to 6 carbon atoms), benzyl, p
nitrobenzyl, p-dimethylaminobenzyl, p-carbome
thoxybenzyl, vinyl, trimethylsilyl, COR8, C02R7,
CON (R8, R9), NO2, S02R8, Po3R8, P02N (R8, R9) and
R7, R8 and R9 are defined as above, can be synthesized from braziliner according to the following reaction scheme

<SEP> OH
<tb><SEP> KO <SEP> there
<tb><SEP> I) o
<tb><SEP> H <SEP> OH
<tb><SEP> OH
<tb><SEP> 8nC1
<tb><SEP> OBrI
<tb><SEP> iÉ <SEP> OH
<tb><SEP> OH
<tb><SEP> R4X <SEP><R3 = <SEP>OR4><SEP><R3 = <SEP>H><SEP>PhOCS> CI
<tb><SEP> I <SEP>
<tb> N <SEP> 0fr '<SEP> OSn
<tb> Eno <SEP> N <SEP> EnO
<tb><SEP> OR4 <SEP> Eno <SEP> i) <SEP> O
<tb><SEP> O% ÉOPh
<tb><SEP> Reflux
<tb> H2 / Pd-C
<tb><SEP> 2 / <SEP> HZ / Pd-C
<tb><SEP> OH <SEP> OH
<tb><SEP> NEED <SEP> N
<tb> HO <SEP> HO <SEP> iÉÂ
<tb> Hv10 "
<tb><SEP> Brazilane
<tb>
In this diagram - Bn represents a protective group for phenolic OHs such as the benzyl group, - R4X 'represents an alkylating agent.

After having protected the 3 phenolic hydroxyls in the form for example of benzyl ether, the tertiary alcohol group is removed in the form of phenylthionocarbonate, for example, or else alkylated using various alkylating agents such as, for example, trimethyloxonium tetrafluoroborate, methyl iodide, benzyl bromide, vinyl chloride, trialkylsilane chloride, or else acylated using various acylating agents such as, for example, acetyl chloride, an alkyl chloroformate, a dialkylcarbamate chloride, or else nitrated, or else phosphorylated with, for example, an alkylphosphonate chloride, or alternatively sulfonated with, for example, an alkylsulfonate chloride such as tosyl chloride.

The desired product is obtained after deprotection by catalytic hydrogenation, in particular in the presence of palladium on carbon, concomitant with a reduction in the case of brazilane.

Examples 1 to 3 of the experimental part which follow illustrate the preparation of compounds of this type.

The compounds of the second series which correspond to the general formula I, in which
R1 = R2 = COR5, S02R5, PG2R5, CO2R6, SO3R6 or Po3R6, and
R3 = hydrogen or OR4, with R4 = hydrogen, COR5, S02R5,
P02R5, C02R6, S03R6 or P03R6,
R5 and R6 being defined as above, can be synthesized according to the following general procedure
Brazilin or its derivative (R3 = H), in solution in a freshly distilled solvent, such as for example dichloromethane, is reacted with the acid chloride, such as for example p-nitrosulfonyl chloride, or anhydride acid, such as for example trifluoromethylsulfonic anhydride or 1 trifluoroacetic anhydride, corresponding to the desired ester.

Example 4 of the experimental part which follows illustrates the preparation of a compound of this type in which
R1 = R2 = trifluoroacetyl and R3 = OR4, with R4 = trifluoroacetyl.

The compounds of the third series which correspond to the general formula I, in which
R1 = R2 = COR5, S02R5, PG2R5, C02R6, SO3R6 or Po3R6, and
R3 = OR4,
R4 having the meanings indicated above, with the exception of hydrogen, COR5, SO2R5, Po2R5, C02R6, SO3R6 and Po3R6, and R5 and R6 being defined as above, can be synthesized according to the following general procedure
Braziliner in solution in a freshly distilled solvent, such as for example dichloromethane, is reacted with the acid chloride, such as for example p-nitrobenzenesulfonyl chloride or trifluoromethyl chlorosulfonate, or acid anhydride. , such as for example trifluoromethanesulfonic anhydride or trifluoroacetic anhydride, corresponding to the desired ester. Then, the tertiary alcohol group is alkylated using an alkylating agent, such as trimethyloxonium tetrafluoroborate, methyl iodide, benzyl bromide, vinyl chloride, a trialkylsilane chloride, or else acylated with using an acylating agent, such as for example acetyl chloride, an alkyl chloroformate, an alkyl chloroforamidate, or else nitrated, or else phosphorylated with for example an alkyl phosphochloride, or else sulfonated with for example an alkyl sulfochloride such as tosyl chloride, chosen according to the meaning of R4.

It is also possible to use a brazilin already suitably substituted on the tertiary alcohol group and subject it to the esterification reaction.

The compounds of the fourth series which correspond to the general formula I, in which
R1 = hydrogen, .COR5, S02R5, Po2R5, C02R6, S03R6, or Po3R6,
R2 = alkyl (with 1 to 6 carbon atoms), and
R3 = hydrogen or OR4,
R4, R5 and R6 being defined as above.

can be prepared according to the following general procedure
Brazilin or a derivative thereof, in which
R1 = R2 = hydrogen and R3 is different from OH, is protected on its two catecholic hydroxyls by a methylene bridge, for example by means of formaldehyde. The derivative obtained is alkylated on its unprotected hydroxyl, using an alkylating agent such as for example an alkyl halide, then deprotected in an acidic medium and optionally esterified.

Example 5 of the experimental part which follows illustrates the preparation of a compound of this type in which
R1 = hydrogen, R2 = methyl and R3 = OH.

All these brazilin derivatives are obtained by chemical modification of its hydroxyl groups.

The modifications made make it possible - on the one hand, to modulate the sensitivity of the SOD activity assay (first, third and fourth series), - on the other hand, to have compounds which are stable with respect to autoxidation. , during their storage and which will return the brazilin or a modified brazilin in position 3, less than 20 seconds after their dissolution under the operating conditions of the SOD activity assay (second and third series, as well as fourth series when R1 does not represent hydrogen).

When the determination of the SOD activity must be carried out in a medium containing one or more compounds comprising one or more thiol groups, such as in particular a biological medium, the reducing power of the latter is likely to cause interference which should be determined. 'eliminate.

According to the invention, it has been found that such interferences could be easily avoided by adding to the medium in which the assay is carried out at least one reagent chosen from compounds of pyridinium and quinolinium types, the use of which as "sea scavengers. captans "is the subject or j and of the French patent application filed at the same time in the name of BIOXYTECH and which is entitled:" Mercaptan trapping reagents, their preparation and their applications ".

These reagents meet the following criteria - specificity with respect to mercaptans under the operating conditions used, - rapidity of reaction, - non-interference with the other reagents used in the determination of the SOD activity.

dans laquelle ::
z1 = alkyle (avec 1 à 6 atomes de carbone), benzyle, p
nitrobenzyle, phényle, o,p-dinitrophényle,
-CH2-COOH ou -CH2-CH2-COOH;
Z4 = hydrogène et
Z5 = hydrogène ou alkyle (avec 1 à 6 atomes de
carbone) ou
Z4 et Z5 forment ensemble, avec les deux atomes de
carbone intermédiaires, un cycle phényle;
X = halogénure, sulfonate, fluorosulfonate, phospho
nate, tétrafluoroborate ou tosylate; et soit z2 = vinyle, et
Z3 = hydrogène ou alkyle (avec 1 à 6 atomes de carbone)
(composés 2-vinyliques), soit Z3 : vinyle, et
z2 = hydrogène ou alkyle (avec 1 à 6 atomes de carbone)
6composés 4-vinyliques).More precisely, according to another of its aspects, the subject of the invention is a method for assaying superoxide dismutase (SOD) activity in a liquid medium containing one or more mercaptan (s), in particular a biological medium, which method uses the activation of the autoxidation of a reagent by the SOD activity, characterized in that the said reagent is a compound corresponding to the general formula I defined above and in that the medium also contains an amount capable of completely trapping said mercaptans by S-alkylation of at least one compound corresponding to the general formula II

in which ::
z1 = alkyl (with 1 to 6 carbon atoms), benzyl, p
nitrobenzyl, phenyl, o, p-dinitrophenyl,
-CH2-COOH or -CH2-CH2-COOH;
Z4 = hydrogen and
Z5 = hydrogen or alkyl (with 1 to 6 atoms of
carbon) or
Z4 and Z5 together form, with the two atoms of
intermediate carbon, a phenyl ring;
X = halide, sulfonate, fluorosulfonate, phospho
nate, tetrafluoroborate or tosylate; and let z2 = vinyl, and
Z3 = hydrogen or alkyl (with 1 to 6 carbon atoms)
(2-vinyl compounds), or Z3: vinyl, and
z2 = hydrogen or alkyl (with 1 to 6 carbon atoms)
6 4-vinyl compounds).

These compounds are in part known or can be synthesized by analogy to methods used for the synthesis of known compounds.

Thus, 2-vinyl compounds can be synthesized as follows.

The initial derivative (pyridine or substituted quinoline) is subjected to the Comins method (reference 15), using vinyl-magnesium bromide. The compound thus obtained is oxidized with, for example, tetrachloro-1,4-benzoquinone (parachloranil) or sulfur Sg. The alkylation of heterocyclic nitrogen is carried out using an alkylating agent, for example trimethyloxonium tetrafluoroborate, methyl sulfate, benzyl bromide, fluorodinitrobenzene, sodium iodoacetate or 3-bromopyruvate sodium, chosen according to the meaning of Z1.

Example 6 of the experimental part which follows illustrates the preparation of a compound of this type.

On the other hand, 4-vinyl compounds can be synthesized as follows.

The corresponding heterocyclic aldehyde (aldehyde function, -CHO, in position 4) is subjected to the reaction of
Wittig, with, for example, triphenylmethylphosphonium bromide, or the Peterson reaction, with, for example, chloromethyltrimethylsilane. The alkylation of heterocyclic nitrogen is carried out using an alkylating agent, for example trimethyloxonium tetrafluoroborate, methyl sulfate, benzyl bromide, fluorodinitrobenzene, sodium iodoacetate or sodium 4bromopyruvate, chosen according to the meaning of Z1.

Example 7 of the experimental part which follows illustrates the preparation of a compound of this type.

The process according to the invention, in particular when it uses at least one “mercaptan scavenger” defined above, makes it possible to measure the SOD activity of any catalyst of the SOD type in aqueous medium, using a single UV / visible absorption measurement. Its simplicity of implementation is clearly superior to that of the spectrophotometric methods previously described.

This new assay process therefore constitutes a tool of choice for biological research in general as well as in clinical chemistry, in particular for the development of assay kits or kits.

In the latter case, this assay is likely to provide medically useful information in pathophysiological conditions such as diseases with an inflammatory or ischemic component, states of shock, infectious states, systemic diseases and autoimmune diseases, tumors, states of malnutrition, diseases associated with aging, degenerative diseases, hemolytic anemias and syndromes of intoxication by environmental pollutants or by drugs.

Human samples to which the assay can be applied include erythrocytes, blood plasma, platelets, white blood cells, synovial fluid, cerebrospinal fluid, urine or any tissue extract.

In addition, this assay method can be used to carry out the pharmacokinetic studies necessary for the development and use of new drugs exhibiting SOD activity.

This assay method can also be used for the quality control of cosmetic or pharmaceutical preparations exhibiting SOD activity.

In general, this assay method can be used by researchers on any biological sample or any artificial solution capable of exhibiting SOD activity.

According to a preferred embodiment, the method for assaying the superoxide dismutase activity of a sample, in particular biological, is based on a spectrophotometric measurement of the autoxidation rate of a compound of general formula I above, in l 'absence and in the presence of said sample.

More precisely, according to a preferred embodiment, the subject of the invention is a method for assaying the superoxide dismutase (SOD) activity in a liquid sample, characterized in that it essentially comprises the steps consisting in 10) determining the maximum rate of autoxidation Vs of a compound of general formula I in the presence of the sample, by means of the change in absorbance as a function of time, at the wavelength characterizing the appearance of the product of autoxidation of the compound of general formula I used, in a reaction medium buffered to a pH value of 8.0 to 9.0, initiating the reaction by adding an aliquot of a stock solution of the compound of general formula I 20) determining, under the same conditions, the maximum rate of autoxidation Vc of the same compound in the absence of the sample; and 30) determining the SOD concentration of the sample by means of the difference between the measurements taken and a calibration curve established under the same operating conditions.

The reaction medium is advantageously constituted by an amino buffer, such as for example 2-amino-2methyl-1,3-propanediol (AMPD), containing 0.1 mM diethylenetriaminepenta-acetic acid (DTPA) and making it possible to bind the pH at a value in the range from 8.0 to 9.0, at the measurement temperature, for example by adding sodium hydroxide or hydrochloric acid, depending on the nature of the buffer considered.

This reaction medium must be equilibrated at the working temperature and saturated with air at this temperature.

The chosen working temperature, for example 370C, must be kept constant during the measurements.

The reaction is started by adding to the reaction medium (with or without SOD) an aliquot of a stock solution of compound of general formula I in an aqueous solution or in an organic solvent miscible with water, such as, for example, acetonitrile, dimethylformamide or dimethylsulfoxide. In the case of brazilin or one of its derivatives of the first or fourth series, defined above, this stock solution must be prepared anaerobically, except, as described below, if a derivative boron is used in the preparation of this solution.

After homogenization of the solution, the change in absorbance is recorded as a function of time, at the wavelength characterizing the appearance of the autoxidation product of the reagent used (539 nm, for example, for brazilin), in order to to determine the maximum rate of autoxidation.

The determination of the SOD activity in a biological sample is carried out by determining the difference in the rates measured in the presence (Vs) and in the absence of the sample (VC). This difference can be reported for example on the ordinate of a calibration curve which was established under the same operating conditions from, for example, an SOD standard.

This calibration curve can be plotted by representing, as a function of the concentration of the standard used, the variation of the difference in the rate of autoxidation measured in the presence and in the absence of this standard.

However, according to a preferred embodiment, this calibration curve is plotted by representing the variation of the inverse of the difference in the speeds in question, as a function of the inverse of the standard concentration.

Depending on the intensity of the SOD activity to be measured, the sensitivity of the assay in the pH range used (8.0 to 9.0) can be adapted by modifying, for example, one or more of the following parameters: concentration of compound of general formula I selected, buffer concentration and measurement temperature.

Boric acid is an example of a boron derivative which can be used to also modulate the rate of autoxidation of the compound of general formula I used, with the aim of reducing this rate.

In addition, if the stock solution of a compound of general formula I of the first or fourth series, defined above (unprotected catecholic OH) is prepared in the presence of such a boron derivative, it is possible to dispense with an anaerobic preparation of the stock solution of the reagent for use for a given time, which depends in particular on the respective concentrations of the reagent and of the boron derivative, on the nature of the latter and on the storage conditions of the stock reagent solution. Thus, an aqueous solution of brazilin prepared with boric acid, such that the ratio of the concentrations of boric acid and brazilin is equal to 75, is stable for at least one month in aerobiosis, if the solution is stored between 0 and 40C .

The existence, in a sample, in particular biological, of compounds interfering with the assay is most often manifested by a decrease in the speed measured in the presence of the sample compared to that measured in its absence.

In the case of interference linked to the presence of mercaptans, such as for example glutathione, the introduction into the reaction medium of a “mercaptan trapping reagent” as defined above is then necessary.

Thus 1,4,6-trimethyl-2vinylpyridinium tetrafluoroborate (compound of Example 6), for example, will be incorporated at a concentration fifty times greater than that of glutathione for the determination of the SOD activity in an erythrocyte lysate.

If the existence, in a sample, in particular biological, of a compound capable of introducing an artefactual activation of the autoxidation of the compound of general formula I is suspected, a combination such as that described below, which involves a second measurement, carried out under conditions of inhibition of autoxidation, should be undertaken.

Likewise, in the case of the exploration of media, in particular biological, exhibiting low SOD activity, the sensitivity and precision of the assay can be increased by combining the measurement made under conditions of activation of autoxidation, a measurement carried out under conditions of inhibition of autoxidation.

A subject of the invention is therefore also a method of the type described above, characterized in that it also comprises a measurement carried out under conditions of inhibition of autoxidation.

The reaction medium used for this second measurement differs from the medium described above by the use of a buffer at a pH in the range of 7.2 to 7.8, in particular a phosphate buffer consisting, for example, of an adequate mixture of potassium dihydrogenphosphate, dipotassium hydrogenphosphate, all other things being equal.

The maximum rate of autoxidation of the compound of general formula is measured under these operating conditions.
I used in the absence (V ′ C) and in the presence of the sample (V ′). The determination of the SOD activity in the sample is then obtained by means of (VS - VC - V'S + V'C).

As previously, the value obtained can be plotted on the ordinate of a calibration curve, which was established under the same experimental conditions.

The new assay method according to the invention using brazilin or one of its derivatives of general formula I is, by its principle, a specific method of measuring the SOD activity. It is sensitive, fast and easy to use, and is furthermore fully automatable; it is therefore suitable for processing large series of samples, as can be the case in chemistry
Clinical.

In addition, the interferences linked to the presence of mercaptans, frequently encountered in biological samples, are easily eliminated by the use of at least one "mercaptan scavenging reagent" as defined above, without any additional phase of treatment. sample.

Finally, the very flexible operating conditions of this assay method give it flexibility of use allowing it to be adapted to very diverse samples, in particular biological samples, and therefore to take into account the possible particular needs of certain users, as is the case. the case in research.

Examples of application of the new method for assaying the SOD activity using brazilin and three of its derivatives in the case of an erythrocyte lysate are given, by way of illustration, in the experimental part which follows.

The subject of the invention is also a kit or kit for implementing the method for assaying SOD according to the invention.

This kit or kit essentially comprises, as reagent, a compound of general formula I, as defined above in its broadest form.

According to an advantageous embodiment, the kit or kit for carrying out the method for assaying the SOD activity according to the invention further comprises one or more compound (s) scavenger (s) of mercaptans, of general formula II, as defined above.

According to a preferred embodiment, the subject of the invention is also a kit or kit for carrying out the method for assaying the SOD activity according to the invention, characterized in that it essentially comprises - a compound of formula general I in solution or in
powder, in the presence of boric acid or a derivative of this
last, - one or more compound (s) trapping mercaptans of
general formula II, in solution or powder, and - an AMPD-based buffer, buffering in the pH range
from 8.0 to 9.0.

EXPERIMENTAL PART
I Preparation of brazilin derivatives
Example 1: Preparation of 3-O-methylbraziline Svnthesis of 5 ', 6', 7-tri-O-benzvlbraziline
A solution of brazilin monohydrate (304 mg; 1.0 mmol; ALDRICH) in acetone (2.5 ml) containing benzyl bromide (0.4 ml; 3.36 mmol; JANSSEN) and potassium carbonate anhydrous (1.06 g; 7.68 mmol; OSI), under an inert atmosphere, is heated at reflux for 15 h.

The reaction mixture is purple in color. A sodium hydroxide solution (5 ml, 0.5N) is added, and the solution thus obtained is stirred for 1 h at room temperature. The reaction medium is extracted with ethyl acetate (2x10 ml). The organic phases are combined, washed with water (2x5 ml) and dried over MgSO4. The solvent is evaporated off under reduced pressure. The desired product is purified by chromatography on a MERCK F254 silica column (eluent = hexane / AcOEt, 3/1).

Yield = 530 mg (95.3%).

Physical characteristics
* 1H NMR (200 MHz, CDCl3)
2.45 ppm (s, 1H); 2.83 ppm (d, 1H, J = 16.0Hz); 3.18 ppm
(d, Hr J = 16.0Hz); 3.78 ppm (d, 1H, J = 11.3Hz); 4.02
ppm (d, 1H, J = 11.3Hz); 4.09 ppm (s, 1H); 5.04 ppm (s,
2H); 5.09 ppm (s, 2H); 5.11 ppm (s, 2H); 6.55 ppm (d,
1H, J = 2.6Hz); 6.71 ppm (dd, 1H, J = 2.6-8.5Hz); 6.80 ppm
(s, 1H); 6.88 ppm (s, 1H); 7.17 ppm (d, 1H, J = 8.5Hz);
7.42 ppm (m, 15H).

* MS (IE, 70eV): (M +) = 556 (2), 466 (1), 181 (2), 108 (3), 91 (100)
IE = electronic impact.

Synthesis of 5 ', 6', 7-tri-O-benzyl-3-O-methylbraziline
In a solution of 5 ', 6', 7-tri-O-benzylbraziline (530 mg; 0.95 mmol) in dry DMSO (4 ml), powdered potassium hydroxide (210 mg; 3.80 mmol) is introduced. ), under an inert atmosphere, at room temperature. Methyl iodide (0.12 ml; 1.90 mmol) is then added to the reaction medium which is stirred for 30 minutes under these conditions.

After adding water (5 ml), the solution is extracted with ethyl acetate (15 ml). The organic phase is dried over MgSO4. The solvent is evaporated off under reduced pressure. The desired product is purified by chromatography on a MERCK F254 silica column (eluent = hexane / AcOEt, 2/1).

Yield = 510 mg (94%).

The product is recrystallized from a hexane / AcOEt (3/1) mixture.

Physical characteristics
* pF = 99.5-100.50C.

* 1H NMR (200 MHz, CDCl3)
2.70 ppm (d, 1H, J = 15.8Hz); 3.22 ppm (d, 1H,
J = 15.8Hz); 3.38 ppm (s, 3H); 3.67 ppm (d, 1H,
J = 11.9Hz); 4.20 ppm (s, 1H); 4.26 ppm (d, 1H,
J = 11.9Hz), 5.03 ppm (s, 2H); 5.07 ppm (s, 2H);
5.11 ppm (s, 2H); 6.54 ppm (d, 1H, J = 2.6Hz); 6.68 ppm
(dd, 1Hr J = 2.6-8.5Hz); 6.78 ppm (s, 1H); 6.88 ppm (s,
1H); 7.17 ppm (d, 1H, J = 8.5Hz); 7.42 ppm (m, 15H).

* SM (1Er 70eV): (M +) = 570 (45), (M ± 91) = 479 (12), 91 (100)
Synthesis of 3-O-methylbraziline
A solution of 5 ', 6', 7-tri-O-benzyl-3-O-methyl-braziline (160 mg; 0.28 mmol) in an AcOEt / MeOH (5/2) mixture containing 10% palladium on charcoal (20 mg) is purged for 15 minutes with a stream of hydrogen. The suspension is hydrogenated for 5 h at room temperature with stirring. It is then filtered under an inert atmosphere. The solvent is evaporated off under reduced pressure. The desired product gradually crystallizes.

Yield = 81 mg (96%).

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.68 ppm (d, 1H, J = 15.6Hz); 3.08 ppm (d, 1H,
J = 15.6Hz); 3.28 ppm (s, 3H); 3.52 ppm (d, 1H,
J = 12.1 Hz); 4.03 ppm (s, 1H); 4.24 ppm (d, 1H,
J = 12.1 Hz); 6.28 ppm (d, 1H, J = 2.5Hz); 6.48 ppm (dd,
Hr J = 2.5-8.5Hz); 6.64 ppm (s, 1H); 6.71 ppm (s, 1H);
7.18 ppm (d, 1H, J = 8.5Hz); 7.79 ppm (s, 1H); 7.85 ppm
(s, 1H); 8.43 ppm (s, 1H).

* MS (IE, 70eV): 300 (5), 268 (4), 110 (6), 84 (41), 66 (49).

Example 2: Preparation of brazilan
(R1 = R2 = R3 = H).

Synthesis of 5 '6', 7-tri-O-benzyl-3-O-phenyloxythionocarbonylbraziline
To a solution of 5 ', 6', 7-tri-O-benzylbraziline (280 mg, 0.50 mmol) in 3 ml of THF, cooled to -780C, is added dropwise, under nitrogen and with stirring, a methyllithium solution (0.4 ml of a 1.6 M solution in cyclohexane, JANSSEN). Stirring is maintained for 10 minutes at this temperature, then 1 hour at room temperature. The reaction mixture is again cooled to -780C and phenyl chlorothionocarbonate (0.21 ml, 0.75 mmol, ALDRICH) is added dropwise. The reaction mixture is allowed to come to room temperature and stirred for 1 hour.

The hydrolysis is carried out using ammonium chloride then the product is extracted with ethyl acetate (2x10 ml). The organic phases are combined, washed with saturated sodium chloride solution (2x10 ml), then dried over MgSO4. The solvent is evaporated off in vacuo. The yellow oil thus obtained is chromatographed on a MERCK F254 silica column. The desired product, eluted with an ether / hexane (1/5) r mixture, is obtained with a yield of 83% (280 mg of a colorless solid).

Physical characteristics
* 1H NMR (200 MHz, CDCl3):
3.51 ppm (d, 1H, J = 16.50Hz); 3.71 ppm (d, 1H, J =
16.50 Hz); 3.75 ppm (d, 1H, J = 12.17Hz); 4.58 ppm (s,
1H); 5.05 ppm (s, 4H); 5.09 ppm (s, 2H); 5.27 ppm (d,
1H, J = 12.17Hz); 6.57 ppm (d, 1H, J = 2.56Hz); 6.69
ppm (dd, 1H, J = 2.56-8.45Hz); 6.78 ppm (s, 1H); 6.88
ppm (s, 1H); 7.06 ppm (d, 2H, J = 7.30Hz); 7.19 ppm (d,
1H, J = 8.45Hz); 7.24-7.46 ppm (m, 18H).

* MS (IE, 70eV): (M ± PhOC (OH) = S) = 538 (3); 447 (1.5);
419 (3); 91 (100); 60 (80).

Synthesis of 5 '6', 7-tri-O-benzvlanhydrobraziline
A solution of the thionocarbonate derivative obtained above (100 mg, 0.15 mmol) in 3 ml of dry toluene is heated under reflux for 2 hours. The solvent of the slightly yellowish solution thus obtained is evaporated off in vacuo. The residue is purified by chromatography on a MERCK F254 silica column and eluted with an ether / hexane mixture (1/5). The desired product is obtained with a yield of 82% (64 mg).

Physical characteristics
* mp = 142-1440C (ethyl acetate / hexane, 1/4).

* 1H NMR (200 MHz, CDCl3)
3.30 ppm (s, 2H); 5.05 ppm (s, 2H); 5.12 ppm (s, 2H);
5.17 ppm (s, 2H); 5.21 ppm (s, 2H); 6.58 ppm (s, 1H);
6.60 ppm (dd, 1H, J = 2.50-8.41Hz); 7.13 ppm (s, 1H);
7.30-7.50 ppm (m, 17H).

* MS (IE, 70eV); 538 (12), 447 (3), 419 (6), 91 (100).

Synthesis of brazilan
A solution of the preceding derivative (92 mg, 0.17 mmol) in 5 ml of ethyl acetate is hydrogenated in the presence of palladium on charcoal (10%, 30 mg, ALDRICH) under a pressure of 8 bars for 3 hours at room temperature. The suspension is filtered and the solvent is evaporated off in vacuo. The desired product is obtained after purification by chromatography on a MERCK silica column.
F254 with an ether / hexane eluent (1/1). Yield = 69%.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.47 ppm (dd, 1H, J = 2.38-15.46Hz); 2.80 ppm (m, 1H);
3.03 ppm (dd, 1H, J = 7.32-15.46Hz); 3.47 ppm (t, 1H,
J = 10.62 Hz); 4.02 ppm (dd, 1H, J = 4.48-10.62Hz);
4.06 ppm (d, 1H, J = 7.34Hz); 6.25 ppm (d, 1H, J = 2.52
Hz); 6.45 ppm (dd, 1H, J = 2.52-8.34Hz); 6.67 ppm (s,
1H); 6.80 ppm (s, 1H); 7.20 (d, 1H, J = 8.34Hz); 7.55
ppm (s, 1H); 7.61 ppm (s, 1H); 8.15 ppm (s, 1H).

* SM (IE, 70eV): 270 (100), 253 (31), 240 (18), 205 (22),
161 (39), 115 (25), 101 (24).

Example 3: Preparation of 3-O-terbutyloxy
carbonylbraziline
Synthesis of 5 '6', 7-tri-O-benzyl-3-O-terbutyloxycarbonyl-braziline
To a solution of 5 ', 6', 7-tri-O-benzylbraziline (160 mg, 0.29 mmol) and dimethylaminopyridine (71 mg, 0.58 mmol, JANSSEN) in 5 ml of dichloromethane is added diterbutyldicarbonate (100 W1, 0.44 mmol, ALDRICH) at room temperature, under nitrogen. The reaction mixture is stirred for 24 hours and is then heated under reflux for 18 hours. The solvent is evaporated off in vacuo. The desired product is obtained after purification by chromatography on a MERCK F254 silica column with an eluent: ethyl acetate / hexane (1/4). Yield = 71%.

Physical characteristics
* 1H NMR (200 MHz, CDCl3)
1.43 ppm (s, 9H); 3.24 ppm (d, 1H, J = 16.45Hz); 3.44
ppm (d, 1H, J = 16.45Hz); 3.65 ppm (d, 1H, J = 12.18
Hz); 4.38 ppm (bs, 1H); 4.65 ppm (dd, 1H, J = 1.67-12.18
Hz); 5.06 ppm (s, 2H); 5.09 ppm (s, 4H); 6.50 ppm (d, 1H, J = 2.62Hz); 6.72 ppm (dd, 1H, J = 2.62-8.60Hz); 6.99
ppm (s, 1H); 7.02 ppm (s, 1H); 7.28-7.40 ppm (m, 16H).

* MS (DIC, NH3): (M ++ 18) = 674.

DIC = desorption by chemical ionization.

Synthesis of 3-O-terbutyloxycarbonylbraziline
A solution of the preceding derivative (150 mg, 0.20 mmol) in 5 ml of ethyl acetate is stirred in the presence of palladium on charcoal (10%, 20 mg, ALDRICH) at room temperature under hydrogen pressure (5 bars) for 7 hours. The suspension is filtered and then the filtrate is evaporated to dryness. The desired product is obtained after purification by chromatography on a MERCK silica column.
F254 with an eluent: ethyl acetate / hexane (2/3).

Yield = 61%.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
1.43 ppm (s, 9H); 3.17 ppm (d, 1H, J = 16.45Hz); 3.32
ppm (d, 1H, J = 16.45Hz); 3.60 ppm (d, 1H, J = 12.20
Hz); 4.27 ppm (s, 1H); 4.66 ppm (dd, 1H, J = 1.65-12.20
Hz); 6.33 ppm (d, Hr J = 2.60Hz); 6.54 ppm (dd, 1H, J
= 2.60-8.56Hz); 6.68 ppm (s, 1H); 6.72 ppm (s, 1H);
7.28 ppm (d, 1H, J = 8.56Hz); 7.74 ppm (s, 2H); 8.28
ppm (s, 1H).

* MS (DIC, NH3): (M ++ 1) = 387; (M ++ 18) = 404.

Example 4: Preparation of tetra-3,5l, 6 ', 7-O-
trifluoroacetylbraziline.

Trifluoroacetic anhydride (1 ml, 7.08 mmol, JANSSEN) is mixed with brazilin monohydrate (45 mg, 0.15 mmol; ALDRICH) under an inert atmosphere and at room temperature. The suspension is stirred under these conditions for 20 hours. Over time, the suspension homogenizes. The excess trifluoroacetic anhydride and the trifluoroacetic acid are evaporated to dryness under reduced pressure to yield the desired product.

Physical characteristics
1H NMR (200 MHz, CD3COCD3)
3.70 ppm (d, Hr J = 16.92 Hz); 3.92 ppm (d, 1H, J =
16.92 Hz); 4.02 ppm (d, 1H, J = 13.18Hz); 4.83 ppm (dd,
1H, J = 1.92-13.18Hz); 5.01 ppm (s, 1H); 7.02 ppm (d,
1H, J = 2.44Hz); 7.13 ppm (dd, 1H, J = 2.44-8.52Hz);
7.67 ppm (s, 1H); 7.74 ppm (s, 1H); 7.79 ppm (d, 1H,
J = 8.52 Hz).

Example 5: Preparation of 7-O-methylbraziline
Synthesis of 5 ', 6'-O-methvlenebraziline
A solution of anhydrous potassium fluoride (145 mg, 2.5 mmol, JANSSEN) and brazilin monohydrate (152 mg, 0.5 mmol, ALDRICH) in 1 ml of dimethylformamide is heated and stirred under nitrogen at 1200C. Dibromomethane (39 Al, 0.55 mmol, JANSSEN) is added. After 18 hours of stirring at this temperature, the reaction mixture is cooled to OOC and 4 ml of water are added. The mixture is extracted with ethyl acetate (2x5 ml). The organic phases are combined, washed with water and then with a saturated aqueous solution of sodium chloride and dried over Na2SO4. The solvent is evaporated off under vacuum. The desired product is obtained after purification by chromatography on a MERCK F254 silica column (eluent = ethyl acetate / hexane: 1/1).

Yield: 42%.

The product is recrystallized from an ethyl acetate / hexane (1/1) mixture.

Physical characteristics
* pF = 264.50C (decomposition).

* 1H NMR (200 MHz, DMSO d6)
2.81 ppm (d, 1H, J = 16.3Hz); 2.86 ppm (d, 1H, J = 16.3
Hz); 3.64 ppm (d, 1H, J = 11.8Hz); 3.82 ppm (d, 1Hr
J = 11.8Hz); 3.88 ppm (s, 1H); 5.36 ppm (s, 1H); 5.91
ppm (s, 1H); 5.93 ppm (s, 1H); 6.18 ppm (d, 1H, J = 2.4
Hz); 6.40 ppm (dd, 1H, J = 2.4-8.2Hz); 6.74 ppm (s,
1H); 6.87 ppm (s, 1H); 7.22 ppm (d, 1H, J = 8.20Hz);
9.26 ppm (s, 1H).

* MS (IE, 70eV): 298 (100), 280 (62), 279 (79), 241 (28), 149 (58).

Synthesis of 7-O-methyl-5 ', 6' -0-methylenebraziline:
To a solution of 5 ', 6'-O-methylenebraziline (79 mg, 0.27 mmol) in 2 ml of acetone are added potassium carbonate (183 mg, 1.33 mmol, OSI) and iodide methyl (35 Fl, 0.53 mmol, LANCASTER). The mixture is heated at reflux under nitrogen for 24 hours with stirring. Then, after returning to ambient temperature, the suspension is filtered. The filtrate is evaporated to dryness and the desired product is obtained after purification by chromatography on a MERCK F254 silica column (eluent = ethyl acetate / hexane; 1/4). Yield = 65%.

Physical characteristics
* 1H NMR (200 MHz, CDCl3)
2.58 ppm (s, 1H); 2.80 ppm (d, 1H, J = 16.45Hz); 3.17
ppm (d, 1H, J = 16.45Hz); 3.76 ppm (s, 3H); 3.79 ppm
(d, 1H, J = 11.8Hz); 4.00 ppm (d, 1H, J = 16.45Hz);
4.04 ppm (s, 1H); 5.86 ppm (m, 2H); 6.48 ppm (d, 1H, J =
2.4 Hz); 6.62 ppm (dd, 1H, J = 2.4-8.2Hz); 6.68 ppm (s,
1H); 6.72 ppm (s, 1H); 7.24 ppm (d, 1H, J = 8.20Hz).

Synthesis of 7-O-methylbraziline
To a solution of 7-O-methyl-5 ', 6'-O-methylenebraziline (50 mg, 0.16 mmol) in 2 ml of methylene chloride is added dropwise, under nitrogen and stirring, a solution of trichloride boron (0.25 ml of a 1M solution in 0.25 mmol methylene chloride). The reaction mixture is stirred at room temperature for 20 hours. Absolute methanol (60 Fl) is added slowly and then the solvent is evaporated off in vacuo. The residue is taken up in ethyl acetate (5 ml) and washed with water. The organic phase is dried over Na2SO4 and the solvent is evaporated off in vacuo. The desired product is obtained after purification by chromatography on a silica column.
MERCK F254 (eluent = ethyl acetate / hexane: 1/1).

Yield = 44%.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.80 ppm (d, 1Hr J = 16.5Hz); 3.00 ppm (d, Hr J =
16.50 Hz); 3.70 ppm (s and d, 4H); 3.93 ppm (d, 1H, J =
11.30 Hz); 3.98 ppm (s, 1H); 4.48 ppm (s, 1H); 6.37 ppm
(d, 1H, J = 2.4Hz); 6.56 ppm (dd, 1H, J = 2.4-8.30Hz);
6.63 ppm (s, 1H); 6.77 ppm (s, 1H); 7.26 ppm (d, 1H, J =
8.30 Hz); 7.59 ppm (s, 1H); 7.64 ppm (s, 1H).

* SM (IE, 70eV): 300 (100), 282 (59), 281 (63), 243 (45),
176 (13), 165 (10), 141 (20).

II. Preparation of "mercaptan trappers"
Example 6: Preparation of tetrafluoroborate
1,4,6-trimethyl-2-vinylpyridinium
Synthesis of 4,6-dimethyl-2-vinylpyridine
To a solution of 2,4-dimethylpydirine (20 g; 0.186 mol; LANCASTER), in 200 ml of freshly distilled THF, cooled to -200 ° C. and under an inert atmosphere, is added benzoyl chloride (29.12 g; 0.186 mol. ;
JANSSEN) in 100 ml of THF. The reaction mixture is stirred at this temperature for 20 minutes. Then vinylmagnesium bromide (186 ml of a 1.0 M solution in THF) is added and the solution is stirred at room temperature for 0.5 h. The solvent is evaporated off under reduced pressure and the residue is taken up in 300 ml of terbutylmethyl ether. After washing the reaction medium with a saturated solution of NH4Cl, the organic phase is dried over MgSO4 and the desired product is purified by chromatography on a column of silica MERCK F254 (eluent = hexane / AcOEt, 9/1).

Yield = 39.5 g (83%), colorless oil.

The above product is taken up in 500 ml of toluene and added with p-chloranil (tetrachloro-1,4-benzoquinone) (41.7 g; 0.169 mol), then heated at reflux for 2 h. The reaction medium is concentrated under reduced pressure, then added with a sodium hydroxide solution (260 ml, 2N). The product is extracted with terbutylmethyl ether (300 ml) and purified by chromatography on a MERCK F254 silica column (eluent = hexane / AcOEt, 9/1)
Yield = 11.1 g (54.2%), pale yellow oil.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.20 ppm (s, 3H); 2.50 ppm (s, 3H); 5.25-5.45 ppm (dd,
2H, J = 11.9-18.7Hz); 6.60-6.90 ppm (m, 3H).

* MS (DIC, NH3): MH + = 134 (100).

DIC = desorption by chemical ionization.

Synthesis of 1,4,6-trimethyl-2 vinylpvridinium tetrafluoroborate:
To a solution of 4,6-dimethyl-2-vinylpyridine (0.61 g; 4,13.10-3 mol) in 4.0 ml of methylene chloride under an inert atmosphere is added, in one portion, trimethyloxonium tetrafluoroborate ( 0.50 g; 3.76.10-3 mol). The suspension is left to react for 2 h at 250C.

The suspension is filtered and the product is dried under vacuum.

Yield = 0.162 g (18%), white crystals.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.60 ppm (s, 3H); 2.85 ppm (s, 3H); 4.20 ppm (s, 3H);
6.00 ppm (d, 1H, J = 12.8Hz); 6.25 ppm (d, 1H,
J = 19.2Hz); 7.27 ppm (dd, 1H, J = 12.8-19.2Hz); 7.82 ppm
(s, 1H); 7.95 ppm (s, 1H).

* MS (IE, 70eV): (M +) 148 (10); 147 (18); 133 (34);
121 (100); 106 (25); 91 (31); 77 (52); 49 (77).

* Microanalysis
Calculated (%): C 51.10; H 6.00; N 6.00
Found (%): C 49.80; H 6.08; N 5.96.

Example 7: Preparation of tetrafluoroborate of 1
methyl-4-vinylquinolinium
Synthesis of 4-vinylquinoline
A solution of 4-quinolinecarboxaldehyde (5 g; 3,18.10-2 mol) in 32 ml of benzene, under an inert atmosphere, containing triphenylmethylphosphonium iodide (26 g; 6.36.10-2 mol) is added to a solution sodium hydroxide (96 ml; 5N). The two-phase system is stirred at 450C for 30 minutes. After decantation, the aqueous phase is extracted with ethyl acetate (3x100 ml). The organic phases are combined and washed with a 1N HCl solution (2x30 ml), then the aqueous phase is neutralized with NaHCO3 and extracted with methylene chloride (3x100 ml). The organic phase is dried over MgSO4. The desired product is purified by chromatography on a MERCK F254 silica column (eluent = cyclohexane / AcOEt, 8/2).

Yield = 2.8 g (60%).

Physical characteristics
* 1H NMR (200 MHz, CDCl3)
5.60 ppm (d, 1H, J = 10Hz); 5.95 ppm (d, 1H,
J = 14Hz), 7.30-7.55 ppm (m, 4H); 7.60-7.75 ppm (t, 1H,
J = 4Hz), 8.07 ppm (t, 2H, J = 6Hz); 8.85 ppm (d, 1H,
J = 4 Hz).

Synthesis of 1-methyl-4-vinyl quinolinium tetrafluoroborate
To a solution of 4-vinylquinoline (0.72 g; 4.64.10-3 mol) in an acetonitrile / acetone (2.5 / 1) mixture is added under an inert atmosphere, in one portion, trimethyloxonium tetrafluoroborate (1, 58 g; 10.7.10-3 mol). The suspension is left to react for 1 h at 250C.

The solvent is evaporated off under reduced pressure, the residue is taken up in 2-propanol (25 ml) and then stirred for 30 minutes at room temperature. After addition of ethyl ether (25 ml), the suspension is filtered. The desired product is dried under vacuum.

Yield = 0.93 g (60.4 t).

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
4.30 ppm (s, 3H); 6.25 ppm (d, 1H, J = 12.5Hz); 6.65 ppm
(d, 1H, J = 17.5Hz); 7.85-8.00 ppm (dd, 1H, J = 12.5
17.5 Hz); 8.10 ppm (d, 1H, J = 7Hz); 8.35 ppm (dd, 2H,
J = 10-10 Hz); 8.57 ppm (d, 1H, J = 10Hz); 8.72 ppm (d,
1H, J = 10Hz); 9.35 ppm (d, 1H, J = 7Hz).

* MS (FAB) glycerol-NOBA: positive ion M + = 170 (100)
negative ion M- = 87 (100).

FAB = fast atom bombardment (Fast Atom
Bombardment).

NOBA = meta-nitrobenzyl alcohol.

* Microanalysis
Calculated (%): C 56.00; H 4.60; N 5.40.

Found (%): C 55.56; H 4.35; N 4.12.

III. Examples of applications
The operating protocols described below are examples of application of the assay method using respectively brazilin, brazilan, 3-O-methylbraziline and tetra-3,5 ', 6', 7-O-trifluoroacetylbraziline to measure SOD activity in erythrocyte lysate.

Example 8: Assay procedure using
brazilin
A box containing the reagents described below is used. This reagent kit should be stored at a temperature between 0 and 40C.

Description of reagents R1: 2.10-3 M brazilin solution in the mixture
DMSO / H2O, 50/50 (v / v) containing boric acid
0.15 M.

(Keep between 0 and 40C during measurements; stable
about 1 month after preparation, under the conditions
storage space mentioned above)
R2: 1,4,6-trimethyl- tetrafluoroborate solution
2-vinylpyridinium 5.10-2 M in DMSO containing 25%
(w / v) ethylene glycol.

(Keep between 0 and 40C during measurements; stable
at least 1 month after preparation).

E: Absolute ethanol / chloroform, 62.5 / 37.5 (v / v).

(Store between 0 and 40C for the preparation of
sample).

T: 0.05 M AMPD / HCl buffer pH = 8.8, containing DTPA
0.1 mM.

(Let equilibrate in air and at 370C before measurements)
Preparation of the sample
At least 100 μl of whole blood are centrifuged at 40 ° C. and 3000 rpm for 10 minutes. The supernatant is removed.

The erythrocyte pellet is washed with 0.9% sodium chloride solution (cooled between 0 and 40C) and then centrifuged at 40C and 3000 rpm for 10 minutes. The supernatant is removed. The erythrocyte pellet is taken up with 4 times its volume of distilled water cooled between 0 and 40C and mixed.

To 125 μl of this erythrocyte lysate at 1 / 5th, 200 Wl of mixture E (cooled between 0 and 40C) are added. Mix and centrifuge at 40C and 3000 rpm for 10 minutes. The supernatant thus obtained, freed of hemoglobin, will be used for the assay (it must be stored at 40 ° C. for the measurements).

Dosing procedure:
The measurements are carried out at 370C in a spectrophotometer, the measuring cells of which are thermostatically controlled.

The reaction mixture corresponding to each assay is prepared extemporaneously according to the proportions given in the table below for 1 ml tanks.

Each reaction is triggered by the addition of 10 Wl of reagent R1. After homogenization of the medium, the change in absorbance is recorded immediately and followed for 1 min 30 s.

The absorbance measurement is carried out at 539 nm in cuvettes with 1 cm optical path, against air.

For each measurement, the reaction medium defined in the table below must be incubated for 1 minute at 370C before adding the reagent R1.

<tb>

<SEP> TEMPO <SEP> IONS <SEP> SAMPLES
<tb><SEP> R2 <SEP> 20 <SEP> pl <SEP> 20 <SEP> Rl <SEP>
<tb> Ethanol <SEP> to <SEP> 50 <SEP>%
<tb> (v / v)
<tb> in <SEP> water <SEP> 40 <SEP> pl <SEP>
<tb> distilled
<tb> SAMPLES <SEP> 40 <SEP> fl <SEP>
<tb><SEP> T <SEP> 930 <SEP> Rl <SEP> 930 <SEP> Rl <SEP>
<tb>
For each of the measurements, the maximum rate of autoxidation, expressed in absorbance units per minute, is determined by subtracting the absorbance value recorded after 20 s from that recorded after 80 s, which corresponds to a linear oxidation phase of brazilin.

Expression of results
Under the conditions of this assay, one unit of SOD activity (U) corresponds to an increase in the formation of brazilein (autoxidation product of brazilin), relative to the control, of 0.5 mol.l-1. min-1.

Let VC and Vs be the speeds corresponding respectively to the control and to the sample, the SOD activity of the latter is calculated by plotting the inverse of the difference (Vs-Vc) in
ordinate of the calibration curve shown on the
attached figure 1, in which the inverse of the
SOD concentration, expressed in ml / U, is shown in
abscissa and the inverse of the difference (Vs-Vc), ex
awarded in min / change in absorbance (min / abs.),
is carried on the ordinate;
20 by determining the value of the corresponding abscissa
on this calibration curve; and
30 by multiplying the inverse of the value of this
abscissa by the dilution factor, i.e. here 250,
to express the results in U / ml erythrocytes.

The results can also be expressed by
example in U / ml of whole blood or in U / mg of protein
erythrocytes.

Remarks
The control of the assay procedure can be carried out by assaying the SOD activity of a 1 / 5th erythrocyte lysate, prepared as described above and stored at -700 ° C. in the form of aliquots to serve as a control.

SOD activity is stable for at least 6 months at -700C.

The assay should be performed on a thawed aliquot extemporaneously.

EXAMPLE 9 Assay procedure using brazilane (compound of 1, example 2)
The reagent kit used contains the same reagents as that of Example 8, except that the reagent R1 is replaced by the reagent D1 defined below Dl: 2.10-3 M brazilane solution in the mixture
DMSO / H2O, 50/50 (v / v) containing boric acid
0.15 M. (storage conditions as in
example 8).

Preparation of the sample
As in example 8.

Dosing procedure
The procedure is as in Example 8, except that each reaction is triggered by the addition of 10 Al of reagent D1 and that the change in absorbance is followed for 1 min at 520 nm instead of 1. min 30 s at 539 nm and that the maximum rate of autoxidation, expressed in absorbance units per minute, is obtained by subtracting the absorbance value measured after 20 s from that measured after 50 s (instead of of 80 s), which corresponds to a linear oxidation phase of brazilane.

Expression of results
It is obtained as in Example 8, using the calibration curve shown in Figure 2 attached.

Example 10: Assay procedure using 3-O
methylbraziline
The reagent kit used contains the same reagents as in Example 8, except that R1 is replaced by D2 defined below.

D2: Solution of 3-O-methylbraziline 2.10-3 M in the
DMSO / H2O mixture, 50/50 (v / v) containing acid
boric 0.15 M.

(Keep between 0 and 40C during measurements;
stable for at least 1 month after preparation, within
storage conditions mentioned below).

This box must be stored at a temperature between 0 and 40C.

Preparation of the sample
The preparation of the sample is identical to that described in Example 8.

Dosing procedure
Absorbance measurements are taken at 541 nm.

With this exception, the assay procedure is identical to that described in Example 8.

The expression of the results is identical to that described in Example 8 using a calibration curve specific for 3-O-methylbraziline.

Example 11: Assay procedure using tetra-3,5l, 6l, 7-O-trifluoroacetylbraziline
The reagent kit used contains the reagents R2 and E of that used in Example 8. The reagents R1 and T are replaced respectively by the reagents D3 and T1 described below.

This reagent kit should be stored at a temperature between 0 and 40C.

D3: Solution of tetra-3,5 ', 6', 7-O-trifluoroacetyl-
2.10-3 M brazilin in acetonitrile.

T1: 0.05 M AMPD / HCl buffer pH = 8.8, containing DTPA
0.1 mM and 1.5 mM boric acid.

(Leave to equilibrate in air and at 370C before measurements)
Preparation of the sample
The preparation of the sample is identical to that described in Example 8.

Dosing procedure
After addition of 10 μl of reagent D3 and homogenization of the reaction medium, the change in absorbance at 539 nm is recorded immediately and followed for 1 minute and 30 seconds.

The assay procedure is otherwise identical to that described in Example 8.

Expression of results
The expression of the results is identical to that described in Example 8, using the same calibration curve.

REFERENCES 1. FRIDOVICH I. (1986), Biological effects of the
radical superoxide; Arch. Biochem. Biophys. 247, 1-11.

2. FRIDOVICH I. (1986), Superoxide dismutases; Adv.

Enzymol. 58, 61-97.

3. FRIDOVICH I. (1989), Superoxide dismutases; year
adaptation to a paramagnetic gas; J. Biol. Chem. 264,
7761-7764.

4. KARLSSON K. and MARKLUND SL (1988), Extracellular
superoxide dismutase in the vascular system of mammals;
Biochem. J., 255, 223-228.

5. CZAPSKI G. and GOLDSTEIN S. (1988), The ubiquiness both
of superoxide toxicity and of the protective role of
superoxide dismutase; in: Oxygen Radicals in Biology
and Medicine, SIMIC MG, TAYLOR KA, WARD JF and
VON SONNTAG C. Ed., Plenum Press, New-York and London,
43-46.

6. FRIDOVICH I. (1982), Measuring the activity of
superoxide dismutases: an embarrassment of the rich; in:
Superoxide dismutase Vol. I, OBERLEY LW Ed., CRC Press,
Boca Raton, Florida, 69-77.

7. FLOHE L. and OTTING F. (1984), Superoxide dismutase
assays; Meth. Enzymol., 105, 93-104.

8. BANNISTER JV and CALABRESE (1987), Assays for
superoxide dismutase; in: Methods of Biochemical
Analysis, Vol. 32, John Wiley & Sons, .279-311.

9. FLOHE L. BECKER R., BRIGELIUS R., LENGFELDER E. and
OTTING F. (1989), Convenient assays for superoxide
dismutase; in: CRC Handbook; Free Radicals and
Antioxidants in Biomedicine, Vol.III, MIQUEL J., WEBER
H. and QUINTANILHA A. Eds., CRC Press, Boca Raton,
Florida, 287-293.

10. GOLDSTEIN S., MICHEL C., BORS W., SARAN M. and CZAPSKI
G. (1988), A critical reevaluation of some assay methods
for superoxide dismutase activity; Free Rad. Biol. Med.,
4, 295-303.

11. MARKLUND SL (1976), Spectrophotometric study of
spontaneous disproportionation of superoxide an ion
radical and sensitive direct assay for superoxide
dismutase; J. Biol. Chem., 251, 7504-7507.

12. MARKLUND SL (1985), Quantitation of superoxide
dismutase: Direct assay with potassium superoxide;
in: CRC Handbook of Methods for Oxygen Radical
Research, GREENWALD RA Ed., CRC Press, Boca Raton,
Florida, 249-255.

13. MARTIN JP, DAILEY M. and SUGARMAN E. (1987), Negative
and positive assays of superoxide dismutase based on
hematoxylin autoxidation; Arch. Biochem. Biophys., 251,
329-336.

14. MARTIN JP (1990), Assays for superoxide dismutase
based on autoxidation of hematoxylin; Meth. Enzymol.,
186 220-227.

15. COMINS DL, STROUD ED and HERRICK JJ (1984),
Regioselective alkylation of Grignard reagents to the 1
phenoxycarbonyl salts of alkyl nicotinates;
Heterocycles, 22 (1), 151-157.

Claims (12)

1.- A method of assaying the superoxide dismutase (SOD) activity in a liquid medium, a method which uses the activation of the autoxidation of a reagent by the SOD activity, characterized in that the said reagent is a responsive compound to the general formula I

in which R1 = hydrogen, COR5, S02R5, Po2R5, Co2R6, S03R6 or Po3R6, R2 = hydrogen, alkyl (with 1 to 6 carbon atoms), COR5, S02R5, Po2R5, CO2R6, S03R6 or Po3R6, R3: hydrogen or OR4, with R4 = hydrogen, alkyl (with 1 to 6 carbon atoms), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl, p carbomethoxybenzyl, vinyl, trimethylsilyl, COR5, S02R5, Po2R5, C02R6, SO3R6, Po3R6, COR8, C02R7, CON (R8, R9), NO2, S02R8, Po3R8 or P02N (R8, R9), R5 = CF3, CF2R7 or p-nitrophenyl, R6 = hydrogen, CF3, CF2R7 or p-nitrophenyl, R7 = alkyl (with 1 to 6 carbon atoms), benzyler p nitrobenzyl, p-dimethylaminobenzyl or p-carbo methoxybenzyl, R8 = hydrogen, alkyl (with 1 to 6 carbon atoms), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or p-carbomethoxybenzyl, and R9 = hydrogen, alkyl (with 1 to 6 carbon atoms), benzyl, p-nitrobenzyl, p-dimethylaminobenzyl or p-carbomethoxybenzyl. 2.- Method for assaying superoxide dismutase (SOD) activity in a liquid medium containing one or more mercaptan (s), in particular a biological medium, which method uses the activation of the autoxidation of a reagent by the SOD activity, characterized in that said reagent is a compound corresponding to the general formula I defined above and in that the medium also contains an amount, capable of completely trapping said mercaptans by S-alkylation, of at least one compound corresponding to the general formula II

in which Z1 = alkyl (with 1 to 6 carbonate atoms), benzyl, p nitrobenzyl, phenyl, o, p-dinitrophenyl, -CH2-COOH or -CH2-CH2-COOH ;; Z4 = hydrogen and Z5 = hydrogen or alkyl (with 1 to 6 atoms of carbon) or Z4 and Z5 together form, with the two atoms of intermediate carbon, a phenyl ring; X = halide, sulfonate, fluorosulfonate, phospho nate, tetrafluoroborate or tosylate; and let z2 = vinyl, and Z3 = hydrogen or alkyl (with 1 to 6 atoms of carbon); or Z3: vinyl, and z2 = hydrogen or alkyl (with 1 to 6 atoms of carbon). 3.- Method according to claim 1 or 2, characterized in that it is based on a spectrophotometric measurement of the autoxidation rate of a compound of general formula I, in the absence and in the presence of the sample to be assayed . 4.- Method for assaying superoxide dismutase (SOD) activity in a liquid sample, characterized in that it essentially comprises the steps of 10) determining the maximum rate of autoxidation Vs of a compound of general formula I in the presence of the sample, by means of the change in absorbance as a function of time, at the wavelength characterizing the appearance of the autoxidation product of the compound of general formula I used, in a buffered reaction medium at a pH value of 8.0 to 9.0, initiating the reaction by adding an aliquot of a stock solution of the compound of general formula I 20) determine, under the same conditions, the maximum rate of Vc autoxidation of the same compound in the absence of the sample; and 30) determining the SOD concentration of the sample by means of the difference between the measurements taken and a calibration curve established under the same operating conditions. 5.- Method according to any one of claims 1 to 4, characterized in that it comprises the addition of a boron derivative, in particular boric acid, to modulate the rate of autoxidation of the compound of general formula I. 6. A method according to any one of claims 1 to 5, characterized in that an aqueous solution of a compound of general formula I in the presence of a boron derivative, in particular boric acid, is used to stabilize the compound of general formula I outside the measurement conditions. 7. A method according to any one of claims 4 to 6, characterized in that the measurement made under conditions of activation of the autoxidation of the compound of general formula I is combined with a measurement carried out under conditions of inhibition of this autoxidation, in a pH range of 7.2 to 7.8. 8.- Kit or kit for carrying out the method according to any one of claims 1 to 7, characterized in that it essentially comprises, as reagent, a compound of general formula I. 9. A kit or kit according to claim 8, characterized in that it further comprises one or more compound (s) scavenger (s) of mercaptans of general formula II. 10.- Kit or kit according to claim 9, characterized in that it essentially comprises - a compound of general formula I in solution or in powder, in the presence of boric acid or a derivative of this last, - one or more compound (s) trapping mercaptans of general formula II, in solution or powder, and - an AMPD-based buffer, buffering in the pH range from 8.0 to 9.0. 11.- Compounds of general formula I in which R1 to R3 are defined as in claim 1, it being understood that when R1 = R2 = hydrogen, R3 cannot represent OH. 12.- Preparation of the compound of general formula I in which R1 = R2 = R3 = H, or brazilane, characterized in that it essentially comprises the steps consisting in - protect brazilin on its phenolic hydroxyls, - transform the alcoholic hydroxyl into a group allowing its elimination, and - reduce and deprotect the resulting product.